In a region infected with bacteria, a mucose structured community of bacterial cells enclosed in polymer matrix is found. This complex aggregation of bacterial cells is called biofilm or biological film (J Bacteriol 176: 2137-2142, 1994). In the biofilm, a bacterial colony is enveloped by extracellular matrix (mucosal surface) comprising polymer matrix (composed of polysaccharides and polypeptides). That is, biofilm is a complex composed of a solid biological surface. the bacterial colony, and a non-biological surface, the extracellular matrix. Therefore, in this invention, biofilm indicates the entire structure composed of such extracellular matrix and bacterial colony therein. Biofilm is the concept first proposed in the late 1970 by Professor Costerton, Chief of The Center for Biofilm Engineering, Montana State University, USA, which indicates the environment where many bacteria survive covered by extracellular matrix made of viscous materials secreted by bacteria (the bacteria adhered on a solid surface secrete viscous materials such as polysaccharides, etc). Biofilm is found everywhere in nature. Mucous slime found in rock or pond is one example. Biofilm is a small city of bacteria where bacteria communicate and defense themselves from outside world. So, biofilm provides an environment for bacteria to survive under diverse environmental stress including antibiotics.
Biofilm is frequently observed not only in nature but also in relation to infectious disease. It can be formed in organs of human and generated as plaques on teeth and can be generated on medical devices for transplantation or industrial equipments. Therefore, biofilm has been a major concern of researchers who study earache in middle ear and pneumonia accompanied with periodontal disease or cystic fibrosis. According to the report made by NIH, USA in 2002, maximum 80% of total bacterial infection was spread through biofilm.
Even antibiotics effective on planktonic bacteria lose their effect once bacteria form biofilm (Trends Microbiol 9: 34-39, 2001). Once bacteria form biofilm, an antibody cannot invade through the extracellular matrix of biofilm, resulting in disablement of host immune system. One of the best-known of the biofilm-specific properties is the development of antibiotics resistance that can be up to 1.000-fold greater than planktonic cells (Antimicrob Agents Chemother 47: 3407-3414, 2003). The mechanism of increase of resistance against antibiotics by biofilm has not been disclosed but can be outlined by the following three reasons. The first reason is “ecological change of microorganisms”. Once biofilm is formed, adhesion among bacteria becomes strong, so that bacterial colony is not apt to be spread, resulting in the decrease of proliferation. Then, bacteria begin to lose dependence on interaction with environment and accordingly metabolism of bacteria becomes slow and sensitivity against antibiotics decreases.
The second reason is physical properties of “extracellular matrix composed of viscous polysaccharides”. Viscous polysaccharides forming the extracellular matrix have electric property being apt to bind antibiotics. The binding of viscous polysaccharides to antibiotics interrupts the spread of antibiotics. That is, antibiotics cannot be delivered to target bacteria, so that the antibiotics cannot take an effect. The third reason is the “production of an inhibitor”, which is presumably involved in the general antibiotic-resistance acquirement mechanism. The most representative inhibitor inhibiting the effect of antibiotics is β-lactamases produced by Pseudomonas. Once biofilm is formed, bacteria residing therein but not having resistance start acquiring the resistance related genes by horizontal gene transfer and as a result these bacteria turn into resistant bacteria. Once biofilm is generated on infected area, it can be judged the area has become antibiotic-resistant condition. Therefore, once biofilm is generated, it is very difficult to treat infectious disease by using general antibiotics.
Thus, formation of biofilm indicates chronic bacterial infection. As described hereinbefore, sensitivity of bacteria to antibiotics becomes weak, suggesting that normal doses of antibiotics are not effective. To overcome such low sensitivity, antibiotics are over-used, only resulting in production of antibiotic resistant bacteria. That is, bacteria infection, particularly when biofilm is already generated, treatment with antibiotics is not effective any more.
To prevent antibiotics from being disabled by biofilm, a novel antibiotic capable of destroying biofilm is required or a method for co-treatment of a conventional antibiotic and a specific component capable of destroying the extracellular matrix of biofilm has to be developed in order for the conventional antibiotics to be effectively functioning.
Staphylococcus aureus is Gram-positive bacteria, which is a pathogenic microorganism causing purulence, abscess, various pyogenic infection, and sepsis. This is a very dangerous pathogen demonstrating the highest resistance against methicillin (73% at average, which is the top level of resistance world widely), according to the investigation in Korea. That means Staphylococcus aureus that is not killed by methicillin takes 73% by its total population, indicating that Staphylococcus aureus is a very dangerous pathogen. Many strains of Staphylococcus aureus are able to form biofilm. Once biofilm is generated, drug delivery is impossible, resulting in chronic infection. That is, biofilm formation causes chronic infection (FEMS Microbiology Letters 252: 89-96, 2005). The treatment of biofilm-associated disease caused by Staphylococcus aureus is especially difficult, compared with other bacteria infection treatments dealing with biofilms generated by other pathogens. Even if a drug is administered for treating disease, delivery of the drug is difficult because of biofilm. Even if the drug is delivered, the treatment effect on highly resistant Staphylococcus aureus is not so great by the conventional antibiotics based treatment. Therefore, to treat biofilm of Staphylococcus aureus, a novel approach with a novel material is necessary.
Various attempts have been made so far to treat biofilm generated by Staphylococcus aureus. However, the results were not successful. The only effective attempt was using lysostaphin, precisely it was reported that lysostaphin could be useful for removing biofilm generated by Staphylococcus aureus (Antimicrob Agents Chemother 47: 3407-3414, 2003). Lysostaphin is an antibacterial enzyme produced by staphyolococcus that is able to destroy cell wall of staphyolococcus. This enzyme is glycylglycine endopeptidase that specifically digests pentaglycine cross bridges found in peptidoglycanstructure of staphyolococcus. So, lysostaphin is expected as an extremely potent anti-staphylococcal agent. Even if lysostaphin has an excellent anti-bacterial effect, it is not perfect. There are still many staphyolococcuses which are not sensitive to lysostaphin (lysostaphin-resistant strains) (J Clin Microbiol 11: 724-727, 1980; Antimicrob Agents Chemother 47: 3407-3414, 2003). Since lysostaphin sensitivity is different among staphyolococcuses, it cannot be effective in every staphyolococcus. Moreover, lysostaphin resistant strains are being generated. Such lysostaphin-resistant strains are called lysostaphin-resistant Staphylococcus aureus variants (Antimicrob Agents Chemother 51: 475-482, 2007). The mechanism of acquiring resistance against lysostaphin has not been explained, yet. But, there was a report concerning the mechanism saying as follows. When femA gene is mutated and thus nonfunctional FemA protein is expressed, monoglycine cross bridges are generated in peptidoglycan structure, which makes lysostaphin powerless (J Bacteriol 188: 6288-6297, 2006). To overcome the above problem of using lysostaphin, studies have been actively undergoing to establish a method to use lysostaphin together with another enzyme such as lysozyme or antibiotics such as methicillin, oxacillin and vancomycin for better effect (Antimicrob Agents Chemother 21: 631-535, 1982; J Antimicrob Chemother 59: 759-762, 2007; Folia Microbiol (Praha) 51: 381-386, 2006). In spite of co-treatment, if Staphylococcus aureus has a low sensitivity against lysostaphin or resistance, removal of biofilm is still impossible. Therefore, a novel substance is required to overcome the disadvantages of lysostaphin treatment. The novel substance might be administered independently or co-administered with the conventional antibiotics. It will be more preferred if the novel substance can be functioning by different mechanism from lysostaphin or the conventional antibiotics.
The new approach drawing our attention these clays to be able to complement the conventional art is to use bacteriophage. Bacteriophage is a kind of virus-like agent that infects bacteria and is generally called ‘phage’ in short. Bacteriophage is a simple structured organism in which a genetic material composed of nucleic acid is covered with a protein envelope. The nucleic acid is single-stranded or double-stranded DNA or RNA. Bacteriophage was first found by Twort, an English bacteriologist, in 1915 during his study on the phenomenon of melting down of micrococcus colonies as being transparent. In 1917, d'Herelle, a French bacteriologist, discovered that there was something decomposing Shigella disentriae in a filtrate of a dysentery patient's feces and later through his further research he isolated bacteriophage independently and named it as bacteriophage. The term bacteriophage means ‘eating bacteria’. Bacteriophage needs a host for its survival and every bacterium has its specific bacteriophage. Bacteriophage invades into a host and is multiplicated therein. Then, bacteriophage expresses a group of enzymes necessary for decomposing cell wall of a host bacterium. These enzymes destroy cell wall of a host bacterium by attacking peptidoglycan layer involved in rigidity and mechanical strength of cell wall. Such bacteriolytic protein of bacteriophage plays a role in destroying cell wall of a host bacterium to pave the way for bacteriophage to get out of the host. Such bacteriolytic protein of bacteriophage is generally called lysin.
Antibiotics (antibacterial agents) are still major part of the treatment of infectious disease by bacteria. However, since 1980s, excessive use of antibiotics has generated many antibiotic resistant strains and since year 2000, multidrug-resistant strains have been frequently reported. With the recognition of problems of using the conventional antibiotics, studies have been focused on bacteriophage as a highly potent alternative for the conventional antibiotics in many advanced countries. Bacteriophage is not only effective in treatment of antibiotic-resistant strain but also effective in treatment of patients with allergy to antibiotics. It was once reported that lysin was used to kill Bacillus anthracis usable as a biochemical weapon for bioterror (Nature 418: 884-889, 2002). Since then, studies have been actively undergoing to understand lysin having a specific bactericidal activity and its functions.
As an alternative for the conventional antibiotics, bacteriophage and lytic protein derived therefrom also draw our attention as a biofilm remover. There is a description on the use of bacteriophage itself in relation to biofilm (International Publication Number WO 2006/063176 A2; WO 2004/062677 A1). However, bacteriophage has a narrow window of effect, suggesting that one bacteriophage cannot be effective in whole bacteria of one species. So, to secure the effective treatment, diverse bacteriophages are necessary. And if necessary, combination of different bacteriophages might be required. The bacteriophage mixture containing different kinds of bacteriophages is called bacteriophage cocktail. Even among different bacteriophages showing equal effect on the same bacteria, there is a difference in the cleavage site of cell wall peptidoglycan and actual functional mechanisms, producing different results. Therefore, co-use of two different bacteriophages might be more effective than single, separate use of each bacteriophage. 
It has been recently attempted to use lytic protein derived from bacteriophage to remove biofilm. In general, lytic protein derived from bacteriophage exhibits wider spectrum of antibacterial activity than its mother bacteriophage. Therefore, it is expected that lytic protein can be more effective in eliminating biofilm than bacteriophage. However, it seems too early to judge with such a few reports made so far. And, there is no report disclosing the sufficient biofilm removal activity of lytic protein. In relation to the lytic protein derived from bacteriophage, it was once reported that recombinant φ11 endolysin could remove biofilm generated by Staphylococcus aureus (Applied and Environmental Microbiology 73: 347-352, 2007). However, the effect of φ11 endolysin was not sufficient because the antibacterial spectrum was still too narrow. To treat biofilm generated by different Staphylococcus aureus strains, diverse lytic proteins derived from different bacteriophages are required. What we have to keep in our mind herein is that every lytic protein derived from bacteriophage is not capable of removing biofilm. According to the previous reports. φ11 endolysin has biofilm removal activity but φ12 endolysin has not. Therefore, biofilm removal activity is not a common property of lytic protein derived from bacteriophage. So, it is necessary to obtain diverse lytic proteins derived from bacteriophage having biofilm removal activity as well as diverse bacteriophages. 